Immunoassay standards and measurement of clinical biomarkers using intra-assay calibration standards

ABSTRACT

The present invention provides novel compositions and methods for creating quantitative standards to calibrate analytes. These compositions and methods enable the creation of standards and calibrators for analyzing analytes and measuring clinical biomarkers. Also provided are kits comprising the novel compositions for use in assays, for example sandwich immunoassays.

FIELD OF THE INVENTION

This invention relates to novel compositions that can be used asreference standards and calibrators in order to measure clinicalbiomarkers in an immunoassay. This invention also relates to methods ofusing the compositions and kits comprising the compositions.

BACKGROUND OF THE INVENTION

Amyloid Beta (A(β) peptides are generated from the cleavage of AmyloidPrecursor Protein (APP) via beta secretase and gamma secretase enzymaticcomplexes (Wolfe, Biochemistry, 45:7931-7939 (2006)). Beta secretasegenerates the N-terminal ends of these amyloid peptides and gammasecretase generates the C-terminal ends (Wolfe, Biochemistry,45:7931-7939 (2006)). Several species of peptides are subsequentlygenerated, typically ranging from 38 to 42 amino acids in lengthdepending on where the gamma secretase cleaves the APP. Aβ peptides havean extracellular domain (amino acids 1-28) and a transmembrane region(amino acids 29-42) that is embedded in the lipid bilayer. Amyloidpeptides that are 42 amino acids long (Aβ₄₂) are believed to be theputative neurotoxic species, either alone or as aggregates. Theseaggregates are suspected to contribute to the neurodegeneration of thebrain resulting in Alzheimer's disease and dementia. The hypothesis thatAβ₄₂ contributes to clinical dementias is called the amyloid cascadehypothesis as described by Hardy et al. (Science, 256:184-185 (1992)).

One of the characteristics of the Aβ peptides is the ability toself-assemble into oligomers at physiological concentrations (Burdick etal., Journal of Biological Chemistry, 267:546-554 (1992); Cerf et al.,Biochemical Journal, 421:415-423 (2009).). The Aβ₄₂ species is moreprone to forming oligomers as compared to the Aβ₄₀ and Aβ₃₈ species. Themechanism of oligomer formation has been shown to originate from asmall, five amino acid region located at amino acids 16 to 20 (KLVFF)which mediates the binding of Aβ peptides in an anti-parallel manner.This small region has thus been termed the “aggregation domain”(Tjernberg et al., Journal of Biological Chemistry, 271:8545-8548(1996)). Aβ peptide aggregates assemble rapidly (i.e., within minutes)under certain conditions especially at lower pH ranges, with slightlyslower kinetics at neutral or higher pH (Burdick et al., Journal ofBiological Chemistry, 267:546-554 (1992)). The aggregates are poorlysoluble in aqueous solutions, especially in the presence of salts. TheC-terminal ends of the Aβ peptides fold back over the core of the dimervia salt bridges thereby increasing their hydrophobicity and promotingfurther polymerization of the peptides into filaments or fibrils. Theadditional two C-terminal residues in the Aβ₄₂ species providesincreased hydrophobicity in comparison to the other Aβ species (Kim etal., Journal of Biological Chemistry, 280:35069-35076 (2005)).

Clinical data suggests that the degree of dementia and cognitive declinehas a higher correlation with Aβ₄₂ concentration than either the Aβ₄₀ orAβ₃₈ species. This observation, in conjunction with the rapidaggregation properties of Aβ₄₂, has led to the hypothesis thatinhibition of Aβ₄₂ aggregation may have clinical benefits. There havebeen numerous studies showing different mechanisms that can be used toinhibit the formation of Aβ₄₂ aggregates. Tjernberg et al. (Journal ofBiological Chemistry, 271: 8545-8548 (1996)) showed that peptidescomprising the aggregation domain bind well to Aβ peptides and inhibitthe formation of aggregates. Several other molecules that bind to theaggregation domain have also been shown to inhibit amyloid peptideaggregation (Martharu et al., Journal of Neurological Sciences,280:49-58 (2009); Kim et al., Biochemical and Biophysical ResearchCommunications, 303:576-279 (2003)). Substitution of amino acids in theaggregation core domain or the deletion of the entire aggregation domainalso prevents Aβ peptide aggregation and fibril formation (Tjernberg etal., Journal of Biological Chemistry, 274:12619-12625 (1999)). Inaddition, various drugs have been designed to inhibit gamma secretaseactivity in order to lower the amount of Aβ₄₂ and related peptidespecies. The usefulness of these approaches in the clinic is currentlyunder investigation.

In order to assess the effectiveness of a molecule to inhibit thegeneration of Aβ₄₂ or to prevent its aggregation, it is necessary tomeasure the amount of Aβ₄₂ accurately. There are several techniques thatare used to detect and quantitate Aβ₄₂ in biological samples includingboth immunoassays (Olsson et al., Clinical Chemistry, 51:336-345 (2005);Verwey et al., Journal of Immunological Methods, 348:57-66 (2009);Sjogren et al., Journal of Neural Transmission, 107:563-679 (2000)) andmass spectrometry (MS) based methods (Cantone et al., Journal ofNeuroscience Methods, 180:255-260 (2009); Journal of Mass Spectrometry,40:142-145 (2005)). The MS based methods, including those of MALDI-TOFand SELDI-TOF along with liquid chromatography prepared massspectrometry, are able to detect many of the amyloid beta species in abiological sample, but do not presently provide sufficient quantitativevalues that are needed for measuring Aβ₄₂ in clinical samples.

Immunoassay methods are based on a double sandwich immunoassay thatcomprises one antibody that is specific to the N-terminus and a secondantibody that is highly specific for the Aβ₄₂ C-terminus (i.e., does notrecognize other Aβ peptide species). There are two basic versions of theimmunoassays. The first version captures Aβ peptides in biologicalsamples via a solid surface immobilized N-terminal region specificantibody. The Aβ₄₂ specific antibody carrying a tag is added to theimmunoassay in order to complete the antibody sandwich. The secondversion captures Aβ peptides in biological samples via an Aβ₄₂C-terminal region specific antibody immobilized on a solid surface. TheN-terminal region specific antibody carrying a tag is added to theimmunoassay. In either version, the tag incorporated via the secondantibody enables the detection of the complete complex. These assays aremade quantitative by the use of Aβ₄₂ reference standards, which areadded in lieu of biological samples. The resulting signal measured fromthe reference standards are used to generate a standard curve which issubsequently used to quantify the amount of Aβ₄₂ in the biologicalsamples.

Until now, the use of Aβ₄₂ reference standards in immunoassays hasrelied on synthetic, full length Aβ₄₂ peptides which are typicallygenerated with minimal difficulty. However, these peptides have stronghydrophobic properties and, therefore, are not soluble in aqueoussolutions. In addition, the storage and use of Aβ₄₂ as referencestandards presents many issues. As discussed, Aβ₄₂ forms aggregatesrapidly and this formation occurs more readily at room temperatures andneutral pH. Long term storage at low temperatures (below −20° C.) andlow pH helps to minimize aggregation during storage but it does notprevent it. Reconstitution of Aβ₄₂ in buffers that are amenable toimmunoassays can also prove difficult. These solutions are almost alwaysaqueous, buffered at a neutral pH, contain salts, and used at roomtemperature; all the conditions that accelerate Aβ₄₂ aggregation. Aβ₄₂peptides that have aggregated are not useful as reference standards inimmunoassays because of the insoluble precipitates and non-uniformity inboth size and availability to be recognized by either detection orcapture antibodies.

Thus, the present invention fulfills a need in the art by providingmethods useful for generating an Aβ₄₂ peptide or protein construct andcompositions thereof that can be used as a reference standard orcalibrator in an immunoassay or other format to measure the abundance ofAβ₄₂ peptide accurately in a fluid or tissue extract sample.Specifically, the compositions and methods of the present invention areaimed at creating non-aggregating peptide reference standards for Aβ₄₂for use in immunoassay formats. The compositions and methods describedherein have a broad applicability to many other peptides that aredifficult to measure and quantitate.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a composition comprising anN-terminal immunoreactive region, a C-terminal immunoreactive region anda linker region. In another aspect, the invention provides a compositioncomprising an N-terminal immunoreactive region, a C-terminalimmunoreactive region and a linker region, wherein the composition isused as a reference standard in an immunoassay. In one embodiment, theimmunoassay is selected from the group consisting of a sandwichimmunoassay, a single antibody assay, a double sandwich immunoassay anda competition assay. In another embodiment, the composition is selectedfrom the group consisting of a protein, a peptide, a fragment and amodified protein. In one embodiment, the N-terminal immunoreactiveregion binds Aβ₄₂, Aβ₄₀, Aβ₃₈, tau or insulin growth factor receptor 1.In another embodiment, the C-terminal immunoreactive region binds Aβ₄₂,Aβ₄₀, Aβ₃₈, tau or insulin growth factor receptor 1. In anotherembodiment, the linker region is a non-immunoreactive domain. In anotherembodiment, the linker region comprises a linker selected from the groupconsisting of polyethylene glycol, a glutamine residue, an alanineresidue, a lysine residue, a lipid, a globular protein, a nucleic acid(including but not limited to DNA, RNA and PNA) and an alkyl chain.

In another aspect, the invention provides an isolated peptide moleculehaving an amino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9.10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23, 24, 25, 26 or 27.

In another aspect, the invention provides a method of measuring thequantity of an analyte in a biological sample, the method comprising:attaching a reference standard to at least two beads thereby forming afirst bead set and a second bead set, wherein the reference standardcomprises an epitope recognized by a first detection antibody andwherein each bead set comprises a different concentration of thereference standard; attaching a capture antibody specific to the analyteto a third bead set; mixing all of the bead sets together to form asuspension array; applying the biological sample to the suspension arraywhereby the analyte binds to the capture antibody on the third bead set;adding a first detection antibody to the suspension array, wherein thefirst detection antibody binds the reference standard and analyte boundto the capture antibody; measuring a first signal from the firstdetection antibody bound to the reference standard in the first beadset; measuring a second signal from the first detection antibody boundto the reference standard in the second bead set; generating a standardcurve based upon the first and second signals; and quantitating theamount of the analyte in the third bead set by measuring a third signalfrom the first detection antibody and comparing the third signal to thefirst and second signal measurements on the standard curve.

In one embodiment, the reference standard comprises a compositioncomprising an N-terminal immunoreactive region, a C-terminalimmunoreactive region and a linker region. In another embodiment, thereference standard comprises a peptide or modified peptide having anamino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 17, 19, 21, 22, 23, 24, 25, 26 or 27.

In another embodiment, the biological sample is selected from the groupconsisting of blood, serum, plasma, peripheral blood mononuclear cells,peripheral blood lymphocytes, tissue, cerebrospinal fluid and cells. Inanother embodiment, the analyte is selected from the group consisting ofAβ₄₂, Aβ₄₀, Aβ₃₈, tau or insulin growth factor receptor 1.

In another embodiment, the method is performed in a multi-well plate,nitrocellulose filter, glass fiber or on a glass slide. In anotherembodiment, the first signal and second signal is a signal selected fromthe group consisting of phycoerythrin, alexa 532,streptavidin-phycoerythrin and streptavidin-Alexa 532. In anotherembodiment, the reference standard is covalently attached to the bead.In another embodiment, the capture antibody is covalently attached tothe bead. In another embodiment, the covalent attachment is acarbodimide bond.

In another aspect, the present invention provides a kit for conductingan immunoassay to detect Aβ₄₂ peptide, the kit comprising a compositionof the present invention.

BRIEF DESCRIPTION OF THE TABLES

Table 1 shows the desirable physical properties of the Aβ peptides andthe tests used to measure those properties.

Table 2 shows Aβ₄₂ peptide and modified peptide sequences anddescriptions.

Table 3 shows tau peptide and modified peptide sequences anddescriptions.

Table 4 shows the measured concentration of Aβ₄₂ peptide in three humancerebrospinal (CSF) samples.

Table 5 shows a list of the novel Aβ peptides that were characterized.

Table 6 shows a summary of dynamic light scattering (DLS) data.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of a two-sided or sandwich soluble standard.

FIG. 2 shows a calibration curve from an exemplary Aβ₄₂ sandwichimmunoassay.

FIG. 3 shows an exemplary Aβ₄₂ sandwich assay using modified Aβ₄₂peptide standards.

FIG. 3A shows a calibration curve from modified standard 2 (SEQ ID NO:2)and 4 (SEQ ID NO:4).

FIG. 3B shows a calibration curve from modified standard 12 (SEQ IDNO:12) and 13 (SEQ ID NO:13).

FIG. 3C shows a calibration curve from modified standard 14 (SEQ IDNO:14) and 6 (SEQ ID NO:6).

FIG. 4 shows a schematic of the Aβ₄₂ intra-assay bead approach.

FIG. 5A shows the measured Median Fluorescence Intensity (MFI) of 6different Luminex bead sets covalently coupled with differentconcentrations of Aβ 1-40 peptide (SEQ ID NO:15).

FIG. 5B shows 4-PL generated calibration curves from either the Aβ₄₀intra-assay standards (circles) or from native Aβ₄₂ peptides as asoluble standard (triangles), similar to the curve shown in FIG. 2.

FIG. 5C shows the measured Aβ₄₂ peptides in human CSF samples usingeither a calibration curve generated from soluble Aβ₄₂ peptides or fromthe intra-assay Aβ₄₀ standards.

FIG. 6 shows the levels of phosphorylated insulin growth factor receptor1 (IGF-R1) in human peripheral blood mononuclear cell lysates. A 4parameter calibration curve was generated from the MFI values on thedifferent bead sets (FIG. 6A) and used to determine the relative levelsof phosphorylated IGF-R1 in PBMC lysates (FIG. 6B).

FIG. 7 shows DLS data.

FIG. 8 shows circular dichroism analysis.

FIG. 9 shows peptide stability data. The full length Aβ₄₂ and sevenmodified peptides were subjected to stability studies at differenttemperatures for up to 40 days (FIGS. 9A-9F).

FIG. 10 shows a standard curve comparison between full length Aβ₄₂ andseven modified peptides.

FIG. 11 shows the standard curve analysis of full length versus modifiedpeptides.

FIG. 12 shows a CSF sample analysis using full length versus modifiedpeptides.

FIG. 13 shows the structures of the polyethylene glycol spacersincorporated into the modified Aβ₍₁₋₄₂₎ peptides.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein.

This invention relates to novel compositions and methods that can beused as reference standards and calibrators in order to measure clinicalbiomarkers in an immunoassay. This invention also relates to methods ofusing the compositions and kits comprising the compositions.Specifically, the compositions and methods of the present invention areaimed at creating non-aggregating peptide reference standards for Aβ₄₂or tau for use in immunoassay formats.

This invention also relates to kits comprising the compositions of theinvention.

Definitions

As used herein, the term “Aβ” refers to amyloid beta.

As used herein, the term “Aβ₄₂” refers to Amyloid Beta 1-42. “Aβ₄₂”refers to a 42 amino acid length peptide that has an amino acid sequenceas noted in Table 2, SEQ ID NO:1.

As used herein, the term “Aβ₃₈” refers to Amyloid Beta 1-3 8. “Aβ₃₈”refers to a 38 amino acid length peptide that has the sequenceDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG (SEQ ID NO:17).

As used herein, the term “Aβ₄₀” refers to Amyloid Beta 1-40. “Aβ₄₀”refers to a 40 amino acid length peptide that has the sequence as notedin Table 2, SEQ ID NO:15.

As used herein, the term “tau” refers to the native tau proteincorresponding to the amino acid sequence as noted in Table 3, SEQ IDNO:20.

As used herein, the term “antibody” is used in the broadest sense, andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(i.e., bispecific antibodies), and antibody fragments (i.e., Fab,F(ab′).sub.2 and Fv) so long as they exhibit binding activity oraffinity for a selected antigen. “Antibody” can also refer to anantibody or antibody fragments hanging or fused to carrierproteins/organisms such as phage or other display carriers that have thesame properties as isolated antibodies.

As used herein, the term “isolated”, as used herein with reference tothe subject proteins and protein complexes, refers to a preparation ofprotein or protein complex that is essentially free from contaminatingproteins that normally would be present with the protein or complex(i.e., in the cellular milieu in which the protein or complex is foundendogenously). Thus, an isolated protein complex is isolated fromcellular components that normally would “contaminate” or interfere withthe study of the complex in isolation, for instance while screening formodulators thereof. It is to be understood, however, that such an“isolated” complex may incorporate other proteins the modulation ofwhich, by the subject protein or protein complex, is being investigated.

As used herein, the term “isolated” as also used herein with respect tonucleic acids, such as DNA or RNA, refers to molecules in a form whichdoes not occur in nature. Moreover, an “isolated nucleic acid” is meantto include nucleic acid fragments which are not naturally occurring asfragments and would not be found in the natural state.

As used herein, the term “nucleic acid” refers to polynucleotide such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded (such assense or antisense) and double-stranded polynucleotide. “Nucleic acid”can also refer to a peptide nucleic acid “PNA” or an artificiallysynthesized DNA or RNA.

As used herein, the terms “peptides”, “proteins” and “polypeptides” areused interchangeably herein. The term “purified protein” refers to apreparation of a protein or proteins that are preferably isolated from,or otherwise substantially free of, other proteins normally associatedwith the protein(s) in a cell or cell lysate. As used herein, the term“modified peptide” refers a peptide that has been modified relative tothe native sequence of that peptide. For example, a modification mayinclude the removal of a deleterious domain or the addition of a linkerwithin the native peptide sequence.

As used herein, the term “binding” refers to a direct associationbetween two molecules, due to, for example, covalent, electrostatic,hydrophobic, ionic and/or hydrogen-bond interactions under physiologicalconditions. Likewise, “complex formation,” between two or morepolypeptides, refers to a direct association between polypeptides, dueto, for example, covalent, electrostatic, hydrophobic, ionic and/orhydrogen-bond interactions under physiological conditions.

As used herein, the term “domain” refers to a region of a protein thatcomprises a particular structure and/or performs a particular function(i.e., aggregation domain, “phosphorylation domain”). The term“aggregation domain” as used herein refers to a five amino acid regionlocated at amino acids 16 to 20 (KLVFF (SEQ ID NO:18)) which mediatesthe binding of Aβ peptides in an anti-parallel manner.

As used herein, the term “immunoreactive domain” refers to a region of aprotein that comprises a particular amino acid sequence that can berecognized by an antibody. This region includes amino acid sequencesthat contain modifications such as glycosylation, methylation,phosphorylation or any other post-translational modification known toone of ordinary skill in the art. Examples of amino acids that could bephosphorylated are tyrosine, serine, or threonine amino acids. An“immunoreactive domain” that includes amino acids that arephosphorylated would also be characterized as a phosphorylation domain.An “immunoreactive” domain would also include two or more regions of aprotein that are in close proximity to one another in the proteinsnative folded state, which together comprise an antibody binding site.

As used herein, the term “immunoassay” as used herein refers to abiochemical test that utilizes one or more antibodies to measure thepresence or concentration of an analyte in a biological matrix. Thisassay can produce a measurable signal in response to a specific bindingof an antibody to an immunoreactive domain of a specific protein orpeptide.

Reference Standards

In one aspect, this invention relates to compositions that can be usedas reference standards and calibrators in order to measure clinicalbiomarkers. In one embodiment, the reference standard comprises apeptide. The peptide may be a modified peptide. The modified peptide maycomprise a linker, a deletion or substitution in a non-immunoreactivedomain. In another embodiment, the non-immunoreactive domain is anaggregation domain or phosphorylation domain.

Alterations in the Aggregation or Non-immunoreactive Domain

In one aspect, the present invention provides modified peptides whichcan be used as reference standards. There are known domains oramino-acid sequences which lead to the self-aggregation and non-specificinteractions of sticky peptides like Aβ₄₂ with itself and othermolecules. As such, it is possible to construct standards or calibratorswhich lack these deleterious domains. The present invention providesseveral ways in which to modify the peptides in order to remove thedeleterious domains. In one embodiment, the amino acids comprising thedeleterious domains are deleted from the amino acid sequence and theN-terminal immunoreactive domain is connected to the C-terminalimmunoreactive domain, lacking the central 17-20 amino acid sequence aswell as various lengths of adjacent C-terminal peptide in the case ofAβ₄₂. Examples of Aβ₄₂ peptides with the central domains deleted areshown in Table 2, SEQ ID NOs:2, 3, and 4. In another embodiment, thecentral aggregation domain plus the adjacent amino acids are replacedwith a linker or spacer consisting of many different types of matter.

In another embodiment, amino acids that do not aggregate arecontemplated as the linker. In one embodiment, the amino acids that donot aggregate are in the form of a hydrophilic spacer or linker of theamino acid sequence or form EERP, shown with both the C-terminal 37-42sequences of Aβ₄₂ (SEQ ID NO:5) and the C-terminal 32-42 portions ofAβ₄₂ (SEQ ID NO:6). In another embodiment, the Aβ₄₂ peptide includes alonger hydrophilic linker, for example the amino acid sequence DREPNR(SEQ ID NO:16), with both the C-terminal 37-42 (SEQ ID NO:7) andC-terminal 32-42 (SEQ ID NO:8) portions of Aβ₄₂.

In yet another embodiment, a series of charged residues are used in theform of the linker between the N-terminal and C-terminal immunoreactivedomains. In another embodiment, a linker is created consisting of aninteger number m Lysine residues (SEQ ID NO:9) or integer number nGlutamic acid residues (SEQ ID NO:10). In another embodiment, a stringof neutral residues is used as a linker. In another embodiment, aconstruct consisting of an integer number p alanine residues (SEQ IDNO:11) is contemplated.

In another embodiment of the present invention, various forms ofpolyethylene glycol (PEG) are used as a linker. In a preferredembodiment, PEG-6atom and PEG-20atom are used with various C-terminalportions (SEQ ID NOs:12-14). In another embodiment, any polymer withchemistry able to couple to amino acid residues is used as a linker orspacer. This polymer includes those of a linear form as well as those ofknown branched topology, like dendrimers and branched co-polymers, tosimulate the immunoreactivity of oligomers of Aβ₄₂ and other similarsticky or self aggregating molecules.

Phosphorylated regions of Tau may also be used to generate modifiedpeptides that may be used as reference standards. The abnormallyhyperphosphorylated tau is associated with neurofibrillary tangles.There are multiple phosphorylation sites of Tau; each of them hasdifferent effect on its biological function. Measurement ofphosphorylated Tau at Ser202/Thr181/Thr212/Thr231 or Ser262 may help tounderstand which one correlates with cognitive decline in MCI subject.Thus, in another embodiment, a modified tau peptide is contemplated asthe reference standard. Examples of modified tau peptides are shown inTable 3.

In another embodiment, the linker comprises any one of the followingmolecules: lipids, globular proteins, nucleic acids (including but notlimited to DNA, RNA and PNA), alkyl chains, or any other linkage thatadds to the stability of the two immune epitopes of interest in theimmunoassay. In another embodiment, the bond between the peptidebackbone and linker comprises a covalent bond, avidin-biotin complex orany other stable bond. In another embodiment, the construct does notlead to self aggregation or non specific absorption to laboratoryplastics, in particular polypropylene, polystyrene, polycarbon and otherlaboratory plastic resins of which pipette tips, tubes, plates and othervessels which hold fluids in which the analyte of interest can bemeasured.

In another embodiment, the novel Aβ₄₂ or tau immunoassay standards orcalibrators require the presence of the N-terminal epitope that isrecognized by the N-terminal specific antibody. There are manyAβ₄₂N-terminal binding antibodies that are known in the art. Forexample, 6E10 is known to recognize the Aβ₃₋₈ epitope whereas 3D6 isknown to recognize the N-terminal epitope of Aβ. An overlapping epitopemay be designed to allow a selection of several N-terminal specificantibodies to be used, depending on the immunoassay requirements anddetection system.

In another embodiment, the novel Aβ₄₂ immunoassay standards orcalibrators require the presence of the C-terminal epitope that isrecognized by the C-terminal Aβ₄₂ specific antibody. Examples of wellcharacterized C-terminal Aβ₄₂ neo-epitope antibodies include G2-11 (fromHeidelberg University), 21F12 (from Athena Diagnostics), 4D7A3 (fromInnogenetics), and 12F4 (from Covance, formerly Signet). An overlappingc-terminal epitope may be designed to allow a selection of severalC-terminal Aβ₄₂ specific antibodies to be used, depending on theimmunoassay requirements and detection system.

In another embodiment, any N-terminal binding, C-terminal binding orphosphorylated tau binding antibody known to one of ordinary skill inthe art may be used herein.

The invention also relates to methods for generating a peptide orprotein construct and compositions thereof that can be used as areference standard or calibrator in an immunoassay to measure theabundance of a peptide accurately in a fluid or tissue extract sample.An immunoassay often requires biologically specific capture reagents,such as antibodies, to capture the analytes or biomarkers of interest.Antibodies can be produced by methods well known in the art, i.e., byimmunizing animals with the biomarkers as antigens. Biomarkers can beisolated from samples based on their binding characteristics.Alternatively, if the amino acid sequence of a polypeptide biomarker isknown, the polypeptide can be synthesized and used to generateantibodies by methods well known in the art. Examples of biomarkersinclude Aβ peptides and tau.

This invention contemplates traditional immunoassays including, forexample, sandwich immunoassays including ELISA or fluorescence-basedimmunoassays, as well as other enzyme immunoassays. In the SELDI-basedimmunoassay, a biospecific capture reagent for the biomarker is attachedto the surface of an mass spectrometry (MS) probe, such as apre-activated PROTEINCHIP® array. The biomarker is then specificallycaptured on the biochip through this reagent, and the captured biomarkeris detected by mass spectrometry.

Thus, in one aspect, the invention relates to methods of measuring theclinical markers with the reference standards of the invention. In oneembodiment, the reference standard is measured by immunoassay. Inanother embodiment, the immunoassay is a sandwich immunoassay. Inanother embodiment, the immunoassay is a single antibody immunoassay,often run in a competitive or “competition” mode for immunoreactivebinding sites. In yet another embodiment, the immunoassay is a doublesandwich immunoassay or enzyme linked immunosorbant assay (ELISA).

In a preferred embodiment, the method of measuring the clinical markersis by using an immunoassay comprising the steps of attaching a referencestandard to at least two beads thereby forming a first bead set and asecond bead set, wherein the reference standard comprises an epitoperecognized by a first detection antibody and wherein each bead setcomprises a different concentration of the reference standard; attachinga capture antibody specific to the analyte to a third bead set; mixingall of the bead sets together to form a suspension array; applying thebiological sample to the suspension array whereby the analyte binds tothe capture antibody on the third bead set; adding a first detectionantibody to the suspension array, wherein the first detection antibodybinds the reference standard and analyte bound to the capture antibody;measuring a first signal from the first detection antibody bound to thereference standard in the first bead set; measuring a second signal fromthe first detection antibody bound to the reference standard in thesecond bead set; generating a standard curve based upon the first andsecond signals; and quantitating the amount of the analyte in the thirdbead set by measuring a third signal from the first detection antibodyand comparing the third signal to the first and second signalmeasurements on the standard curve. It is understood without limitationto the present invention that a bead set could be replaced by any othersolid phase in which multiplexed information can be measuredindependently in a particular detection technology or instrument.

In one embodiment, the reference standard comprises a compositiondescribed herein. In another embodiment, the biological sample isselected from the group consisting of blood, serum, plasma, peripheralblood mononuclear cells, peripheral blood lymphocytes tissue,cerebrospinal fluid and cells. In yet another embodiment, the analyte isAβ₄₂, Aβ₄₀, Aβ₃₈, tau or insulin growth factor receptor 1.

The analyte and/or reference standard may be bound to a variety ofsurface. A surface could be any solid phase surface to which an antibodyor reference standard can be immobilized by covalent linkage, passiveabsorbance, biotin-strepavidin or any other linkage known to one ofordinary skill in the art. For example, the surface may be a bead,plate, slides, fiber, surface plamon resonance sensors or any solidsurface.

In another embodiment, the method is performed in a multi-well plate,nitrocellulose filter or on a glass slide. In another embodiment, thefirst and second signals are detected by fluorescence. For example, thefirst signal and second signal may be a signal selected from the groupconsisting of phycoerythrin, alexa 532, streptavidin-phycoerythrin andstreptavidin-Alexa 532. In another embodiment, the signal is detected byenzymatic activity (i.e., horseradish peroxidase or alkalinephosphatase), chemiluminescence, radioactivity, infra-red emission,fluorescence resonance energy transfer (FRET) or any other method knownto one of ordinary skill in the art.

In another aspect, the invention comprises a kit for conducting animmunoassay to detect an Aβ₄₂ or tau peptide, the kit comprising areference standard of the invention.

Performance Comparison of Novel Immunoassay Standard or Calibrator toNative Aβ₄₂

The performance of novel immunoassay standards or calibrators shouldhave comparable performance to the native Aβ₄₂ in an immunoassay. Nativefull length Aβ₄₂ peptides may be synthesized using standard solid phasetechniques or they may be purchased commercially from a number ofvendors as a catalog item (Anaspec Inc., American Peptide Company, orInvitrogen Inc.). Standard methods can be used to verify the abundanceof the full length construction from truncated species using massspectrometry techniques such as amino acid analysis that are well knownin the field (Kanu et al., Journal of Mass Spectrometry, 43:1-22 (2008);Bernstein et al., Journal of American Chemical Society, 127:2075-2084(2005); Li et al., Encyclopedia of Analytical Chemistry, Meyers, R. A.,ed., John Wiley & Sons Ltd. (2009)).

By way of example, Table 1 lists the desirable physical properties ofAβ₄₂ peptides and the various methods used to test these properties.These methods can be employed to determine if the properties of thereference standard are comparable to those of the native Aβ₄₂ peptide.

TABLE 1 Property Test Target Range Reference Solubility SDS-PAGE Greaterthan 90% Analy. Biochem., monomeric form 316: 223-231 (2003) Dynamiclight Less than 5% Meth. Enzymology, scattering aggregated peptides 309:429-459 (1999); J. Biol. Chem., 274: 25945-25952 (1999) Non-specificSpike recovery into Recovery between Aβ Immunoassay adsorption CSF orbuffer 80 and 120% Olsson et al., Aβ₄₂ immunoassay Clinical Chemistry,51: 336-345 (2005) Aggregation SDS-PAGE Greater than 90% PNAS, 100:330-335 Western blotting monomeric form (2003); Analy. Biochem., 316:223-231 (2003) Thioflavin T assay Greater than 90% Meth. Enzymology,monomeric form 309: 274-284 (1999) Fibril formation Microscopy Minimallevels of Protein Pep. Letters, (transmission observable 13: 261-270(2006); electron microscopy, oligomers or fibrils J. Am. Chem. Soc.,optical, Microscopy 125: 15359-15365 (optical, atomic (2003) force)Stability at 25° C., Less than 20% CV 2 hrs 25° C. Olsson et al., −20°C., and −80° C. loss of signal 3 months −20° C., 6 Clinical Chemistry,compared to freshly months −80° C. 51: 336-345 (2005); prepared Aβ₄₂Verwey et al., Via Aβ₄₂ Journal of immunoassay Immunological Methods,348: 57-66 (2009)

Use of Modified Reference Standards or Calibrators in Antibody BasedImmunoassays

In another aspect of the present invention, the modified referencestandard is used in a single antibody based assay. In one embodiment,immunoassays containing a single antibody can be used to measure Aβ₄₂ ortau in a biological sample by a competition immunoassay. A singleantibody specific to Aβ₄₂ or tau is immobilized to a solid surface suchas the well of a microtiter plate, a bead, or other immunoassay relevantsurface. The antibody may be covalently linked via many differentmethods such as EDC mediated linkage of carboxyl and amine groups, orvia passive absorbance or through a Protein A or Protein G interface. AnAβ₄₂ or tau competitor is then generated from an Aβ₄₂ or tau standard orcalibrator containing the full length Aβ₄₂ or tau peptide or a modifiedversion that retains the epitope of the capture antibody. The Aβ₄₂ ortau competitor is used to generate competition between the native Aβ₄₂or tau in the biological sample and the binding site (paratope) on theimmobilized antibody. A paratope is a term used to describe the bindingregion on the antibody that recognizes the epitope or immunoreactivedomain on the analyte. The Aβ₄₂ or tau competitor is tagged fordetection purposes. In one embodiment, the tag is an enzyme such ashorseradish peroxidase or alkaline phosphatase. In another embodiment,the tag is a fluorescein such as phycoerythrin. In yet anotherembodiment, the tag is another tag such as biotin or ruthenium. In yetanother embodiment, the tag is a nucleic acid such as DNA, RNA or PNA,where by detection of the antibody is quantitated using sensitivetechnology to detect the nucleic acid flag, such as Polymerase ChainReaction (PCR). A single concentration of the Aβ₄₂ or tau competitor isused in the assay and would be determined based on the ability tocompete with the natural levels of Aβ₄₂ or tau found in biologicalsamples.

In another embodiment, the assay is made quantitative by establishing acalibration curve. In another embodiment, quantitation is performed bymaking a set of Aβ₄₂ or tau standards or calibrators that are either thefull length Aβ₄₂ or tau peptide or a modified version that retains theepitope of the capture antibody. These untagged standards or calibratorsare prepared in either buffer or a biological matrix that does notcontain Aβ₄₂ or tau. In one embodiment, the calibration curve isestablished by mixing one of concentrations of the untagged standards orcalibrators with the tagged Aβ₄₂ or tau competitor to the immobilizedantibody. The resulting signal value from each tested concentration ofuntagged standard or calibrator is used to generate a standard curve;plotting the concentration of the untagged Aβ₄₂ or tau standards orcalibrators versus the resulting signal values. Once a standardquantitative curve is established, an assay is used to determine thelevels of native Aβ₄₂ or tau in biological samples by mixing the taggedAβ₄₂ or tau competitor at the same fixed concentration with thebiological sample. The resulting signal value is plotted on the standardcurve to determine the level of Aβ₄₂ or tau in the biological sample.

In another aspect of the present invention, the modified referencestandard is used in a sandwich based immunoassay.

Use of Modified Standards or Calibrators in Intra-Assay ReferenceStandard Based Immunoassays

In another aspect of the present invention, Aβ₄₂ or tau peptides areincorporated into an intra-assay calibration system. In this approach, amultiplex immunoassay format such as the Luminex bead based system orthe Meso-Scale Discovery ECL plate based system could be used. Peptidescontaining amino acid residues that encompass the antibody bindingepitope of the detection antibody are generated. In one embodiment,these peptides include modifications that enable them to be covalentlycoupled to a solid phase or modifications that increase their solubilityand use in aqueous immunoassays. These peptides are immobilized atdifferent concentrations to the relevant solid phase as defined bymultiplexed immunoassay systems, in order to create a set of welldefined standards from which to create a standard curve.

The measurement of soluble biomarkers in clinical samples is often doneusing double antibody sandwich assays. These assays require twoantibodies that are specific to the biomarker and a technology in whichto detect the captured biomarker using the second “reporter” or“detection” antibody. Protein reference standards are required in orderto make the assay quantitative. These standards are often in the form ofrecombinant proteins; however, they may also be obtained from biologicalsamples. Traditional assay formats for these assays include ELISAtechniques that provide quantitation suitable for the analysis ofclinical samples. However, they are often limited to one biomarker assayper well. Newer technologies have been developed that allow multiplebiomarkers to be analyzed in a single well or reaction vessel. Some ofthe multiplexed technologies utilize antibodies spotted onto a solidsurface such as glass slides or specialized microtiter plates. Anotherapproach is via suspension arrays where the antibodies are bound tolatex beads which are mixed together in solution to form the array.

In one embodiment, suspension array technology is used. In anotherembodiment, the suspension array technology is the Luminex xMAPtechnology. Luminex xMAP technology uses latex beads that contain aratio of two fluorescent dyes. Different bead ‘sets’ are created byaltering the ratio of these two dyes. The beads are mixed together toform a suspension array. The bead mixture is analyzed by an instrumentthat identifies each bead by the fluorescence ratio as it passes infront of a laser. These bead sets have different modifications on theirsurface that are used for the covalent attachment of molecules such asproteins, peptides, antibodies, etc. This allows assay to be performedon the surface of these beads. Assays are quantitated through theincorporation of a third fluorescent label such as phycoerythrin to areporter antibody directed at the analytes of interest. A second laserin the instrument measures the fluorescence of this reporter label asthe beads move through the instrument.

EXAMPLES Example 1 Sandwich Based Assay that can be Used to Measure Aβ₄₂

A schematic of a two-sided or sandwich soluble standard is shown inFIG. 1. Briefly, capture antibodies specific to the C-terminus of Aβ₄₂were immobilized to a solid surface. A biological sample was addedthereby allowing Aβ₄₂ to be captured by the immobilized antibody. Asecond tagged detection antibody was added that was specific to theN-terminus of the Aβ₄₂. The measured signal generated by the taggeddetection antibody was used for quantitation.

An example of a sandwich assay that can be used to measure Aβ₄₂ is shownin FIG. 2. Briefly, a biotin labeled anti-C-terminal Aβ₄₂ antibody (565)was immobilized to a 96 well Meso-Scale Discovery streptavidin coatedplate (MesoScale Discovery Inc., Gaithersburg, Md. ((MSD)). Referencestandard Aβ₄₂ peptides (full length with native sequence) were added todifferent wells. Aβ₄₂ peptides were captured by the immobilized captureantibody. A second ruthenium tagged (Ru) antibody to the N-terminus ofAβ₄₂ (26D6) was added, completing the sandwich. The complex was detectedusing an MSD sector 6000 instrument using ECL. The raw fluorescenceunits (RFU) measured by the instrument were fit to a 4-parameterlogistic model to create a standard curve. In another example, severalmodified Aβ₄₂ peptides are used as a reference standard (Table 2, SEQ IDNOs:2, 4, 6, 12, 13, and 14). The standard curves generated by thesemodified calibrators are shown in FIG. 3.

TABLE 2 AP42 Peptide and Modified Peptide Sequences and DescriptionsDescription of Peptide/Modified Peptide/Modified Peptide SequencePeptide Sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAII native Aβ₄₂ sequenceGLMVGGVVIA (SEQ ID NO: 1) DAEFRHDSGYEVHMVGGVVIA (SEQ ID NO: 2)native Aβ₄₂ amino acids (1-14) (35-42) with no spacer in betweenDAEFRHDSGYEVHHQKGGVVIA (SEQ ID NO: 3) native Aβ₄₂ amino acids (1-16)(37-42) with no spacer in betweenDAEFRHDSGYEVHHQKIGLMVGGVVIA (SEQ ID NO: 4)native Aβ₄₂ amino acids (1-16) (32-42) with no spacer in betweenDAEFRHDSGYEVHHQKEERPGGVVIA (SEQ ID NO: 5)native Aβ₄₂ amino acids (1-16)- EERP-native Aβ₄₂ amino acids (37-42)DAEFRHDSGYEVHHQKEERPIGLMVGGVVIA  native Aβ₄₂ amino acids (1-16)-(SEQ ID NO: 6) EERP-native Aβ₄₂ amino acids (32-42)DAEFRHDSGYEVHHQKDREERPGGVVIA (SEQ ID NO: 7)native Aβ₄₂ amino acids (1-16)- DREERP hydrophilic linker-nativeAβ₄₂ amino acids (37-42) DAEFRHDSGYEVHHQKDREPNRIGLMVGGVVnative Aβ₄₂ amino acids (1-16)- IA (SEQ ID NO: 8)DREPNR hydrophilic linker - native Aβ₄₂ amino acids (32-42)(1-16)-(Lys)m -(37-42) (SEQ ID NO: 9) native Aβ₄₂ amino acids (1-16)- upto 20 Lysine residues- native Aβ₄₂ amino acids (37-42) (1-16)-(Glu)n-(37-42) (SEQ ID NO: 10) native Aβ₄₂ amino acids (1-16)- upto 20 Glutamic acid residues- native Aβ₄₂ amino acids (37-42)(1-16)-(Ala)p -(37-42) (SEQ ID NO: 11)native Aβ₄₂ amino acids (1-16)- up to 20 Alanine residues- nativeAβ₄₂ amino acids (37-42) DAEFRHDSGYEVHHQK-PEG(20-ATOMS)3-native Aβ₄₂ amino acids (1-16)- GGVVIA (SEQ ID NO: 12)(PEG_20)3 linker- native Aβ₄₂ amino acids (37-42)DAEFRHDSGYEVHHQK-PEG(9-ATOMS)6- native Aβ₄₂ amino acids (1-16)-MVGGVVIA (SEQ ID NO: 13) (PEG_9)6 linker- native Aβ₄₂amino acids (35-42) DAEFRHDSGYEVHHQK-PEG(9-ATOMS)5-native Aβ₄₂ amino acids (1-16)- IGLMVGGVVIA (SEQ ID NO: 14)PEG_9)5 linker- native Aβ₄₂ amino acids (32-42) (X-Y)-Linker-(Z-42)Generic: X,Y,Z, Linker to be specified DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIAβ₄₀ native sequence GLMVGGVV (SEQ ID NO: 15)

An immunoassay can also be used to measure tau. Examples of modified taupeptide sequences that can be used as reference standards of the instantinvention are depicted below in Table 3.

TABLE 3 Tau Peptide Sequences and Constructs Description ofPeptide/Modified Peptide/Modified Peptide Sequence Peptide SequenceMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQ native tau sequenceEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 20)EVMEDHAGTYGL-generic linker-GAAPPGQKGQAN native tau amino acids(SEQ ID NO: 21) (9-20)-generic linker- native tau amino acids (156 -167)EVMEDHAGTYGL-generic linker-SGDRSGYSSP native tau amino acids(SEQ ID NO: 22) (9-20)-generic linker- native tau amino acids- (191-200)GAAPPGQKGQAN-generic linker- native tau amino acidsIPAKTPPAPKT_((PO4))PPSSGEPPK (SEQ ID NO: 23) (156-167)-genericlinker- native tau amino acids (171-190); amino acid T¹⁸⁰ isphosphorylated GAAPPGQKGQAN-generic linker- native tau amino acidsREPKKVAVVRT_((PO4))PPKSPSSAK (SEQ ID NO: 24) (156-167)-generic linker-native tau amino acids (221-240); amino acid T231 is phosphorylatedGAAPPGQKGQAN-generic linker-SGDRSGYSSP native tau amino acids(SEQ ID NO: 25) (156-167)-generic linker- native tau amino acids(191-200) IPAKTPPAPKT_((PO4))PPSSGEPPK-generic linker-native tau amino acids SGDRSGYSSP (SEQ ID NO: 26)(171-190)-generic linker- native tau amino acids (191-200); amino acidT180 is phosphorylated SGDRSGYSSP-generic linker- native tau amino acidsREPKKVAVVRT_((PO4))PPKSPSSAK (SEQ ID NO: 27) (191-200)-generic linker-native tau amino acids (221-240); amino acid T²³¹ is phosphorylated

Example 2 Sandwich Aβ₄₂ Assay Measuring Aβ₄₂ in Biological Samples

An example of a sandwich Aβ₄₂ assay measuring Aβ₄₂ in biological samplesis shown in Table 3. Biotin labeled anti-C-terminal Aβ₄₂ antibody (565)was immobilized to a 96 well MSD streptavidin coated plate. Referencestandard Aβ₄₂ peptides (full length with native sequence) or a modifiedAβ₄₂ reference standard (SEQ ID NO:2) were added to different wells.Human CSF samples at different dilutions were placed in different wells.The plate was incubated 2 hours at room temperature to allow Aβ₄₂peptides to be captured by the immobilized capture antibody. A secondruthenium tagged (Ru) antibody to the N-terminus of Aβ₄₂ (26D6) wasadded, completing the sandwich. The complex was detected using an MSDsector 6000 instrument using ECL. The raw fluorescence units (RFU)measured by the instrument were fit to a 4-parameter logistic model tocreate a standard curve. The measured concentrations of Aβ₄₂ in humanCSF samples are shown in Table 3.

TABLE 4 Measured Concentration of Aβ₄₂ Peptide in Three HumanCerebrospinal (CSF) Samples Aβ₄₂ Concentration (pg/ml) CSF CSF Fulllength Aβ Modified Aβ sample dilution 1-42 Peptide xx-42 peptide CSF-11:2 dil Below limit of detection Below limit of detection CSF-1 1:4 dilBelow limit of detection Below limit of detection CSF-2 1:2 dil 26.227.8 CSF-2 1:4 dil 54.0 56.8 CSF-3 1:2 dil 88.4 80.4 CSF-3 1:4 dil134.4  126.4 

Example 3 Aβ Peptide Based Intra-Assay Luminex Assay

FIG. 4 shows a schematic of the Aβ₄₂ intra-assay bead approach. Beadsets coupled with different concentrations of Aβ₄₂ standard peptides (orother native or modified Aβ peptides) are combined with a bead setcoupled with an anti-Aβ₄₂-C-terminal specific capture antibody to form asuspension array. The array is incubated with a biological sample, wherethe Aβ₄₂ peptide in the biological sample is captured by the beadcoupled with anti-Aβ₄₂ capture antibody. A tagged detection antibodyspecific to the N-terminus of the Aβ₄₂ peptide is added to thesuspension array, thereby binding to the captured Aβ₄₂ peptide and alsoto the beads that have Aβ₄₂ peptides coupled to their surface. The MFIvalues obtained from the beads with Aβ₄₂ peptides coupled to theirsurface is used to generate an intra-assay calibration curve. The amountof Aβ₄₂ in the biological sample is determined from the amount ofcaptured Aβ₄₂ on the bead coupled with the anti-Aβ₄₂ capture using theintra-assay standard curve.

Antibodies and Reference Standards

The native full length Aβ₄₂ and full length Aβ₄₀ peptides (SEQ ID NOs:1and 15, respectively) were obtained from American Peptide Company. The116B565.1 mouse anti-human Aβ₄₂ C-terminus antibody and the 26D6-B2-B3mouse anti-human Aβ₄₂ N-terminus antibody were obtained via protein-Gpurification of culture supernatants produced by the relevant BMS ownedhybridoma cell lines.

Phycoerythrin-streptavidin conjugate was obtained from JacksonImmunoresearch (West Grove, Pa.). Tween-20, 1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC), sodium azide, IgGfree Bovine Serum Albumin (BSA), and sodium phosphate were purchasedfrom Sigma-Aldrich Corporation (St. Louis, Mo.). Phosphate bufferedsaline (PBS) was obtained from Mediatech Incorporated (Herndon, Va.).Carboxylated Luminex beads were purchased from Bio-Rad Incorporated(Hercules, Calif.). Phycoerythrin label goat anti-mouse IgG antibody wasobtained from Jackson Immunoresearch (West Grove, Pa.).

Covalent Coupling of Anti-Aβ₄₂ Capture Antibody to Luminex Beads

The 116B565.1 mouse anti-human Aβ₄₂ C-terminus antibody was covalentlycoupled to the surface of carboxylated beads using a two-stepcarbodimide procedure. The beads were washed by centrifuging 1.25×10⁷beads for 5 min at 14000×g 4 ° C. in an Eppendorf 5415D centrifuge(Westbury, N.Y.). The supernatant was carefully removed and another1.25×10⁷ beads were dispensed and centrifuge for 5 min at 14000×g 4 ° C.The supernatant was again carefully removed and resuspended in 800 μl of0.1M sodium phosphate buffer pH 4.8 (activation buffer). The beads werethen vortexed for 15 seconds and sonicated for 15 seconds using a SPERSCIENTIFIC® LTD Ultrasonic Cleaner (Scottsdale, Ariz.). The beads werewashed 2 additional times with activation buffer, and resuspended in 200μl of freshly prepared 5 mg/ml EDC prepared in activation buffer. Thebeads were incubated in a rotator for 20 min RT protected from light. Atthe end of the EDC step, the beads were washed and resuspended in 1000μl of 250 μg/ml capture antibody prepared in PBS and incubated for lhrsRT in a rotator protected from light. The beads were washed andincubated with 1 ml of blocking buffer (PBS, 1% (w/v) BSA, 0.02% (w/v)Tween-20) in a rotator for 1 hr RT protected from light. Finally, thebeads were counted with a hemacytometer, resuspended in blocking bufferat 2×10⁶ beads/ml, and stored protected from light at 4 ° C. until readyfor use.

Surface Testing of Covalent Coupling Efficiency of Aβ₄₂ CaptureAntibodies to Luminex Beads

The presence of capture antibody on the bead surface was confirmed usingsurface testing. 50 μl of assay buffer (PBS, 1% (w/v) BSA, 0.05% (w/v)Tween 20, 0.05% (w/v) azide) containing 2500 beads were added toMillipore filter bottom plate wells (Bedford, Mass.). The beads werewashed by placing the plate over a Millipore vacuum manifold (Bedford,Mass.) to remove the liquid and then resuspended in 100 μl/well of PBSTwash buffer. Finally, the wash buffer was removed from the wells viavacuum and the beads were incubated with 100 μl/well of PE-Goatanti-mouse IgG diluted 1/100 in assay buffer. The plate was incubated ona 96-well plate shaker (Lab Line Instruments, Melrose Park, Ill.) at 300rpm for 30 min RT protected from light. The beads were subsequentlywashed 3 times via vacuum filtration using 100 μl/well of wash bufferand resuspended in 100 μl/well of assay buffer. The MFI of at least 50beads/well was measured using a Luminex¹⁰⁰ instrument obtained fromBio-Rad Laboratories (Hercules, Calif.) running Bioplex manager 4.1.1software. An MFI of at least 20,000 was used to confirm the presence ofusable quantities of antibody on the bead surface.

Covalent Coupling of Intra-assay Aβ₄₀ Peptides to Luminex Beads

Aβ₄₀ native full length peptides (SEQ ID NO:15) were prepared byreconstituting the lyophilized peptide in 2.5 ml PBS to give a finalconcentration of 10 mg/ml. Aβ₄₀ peptides were selected for this assay inlieu of Aβ₄₂ peptides because they still expressed the N-terminal Aβ₄₂epitopes needed for the binding of 26D6 antibodies and the Aβ₄₀ peptideswere more stable in aqueous buffers needed for conjugation. Aβ₄₀peptides were diluted to different concentrations (shown in FIG. 5A)using diluent buffer (PBS, 1% (w/v) BSA, 0.02% (w/v) Tween-20). Eachpreparation of Aβ₄₀ peptide was covalently coupled to the surface of aselected bead set (different bead set for each concentration) using atwo-step carbodimide procedure. Each bead set was washed by centrifuging1.25×10⁷ beads for 5 min at 14000×g 4° C. in an Eppendorf 5415Dcentrifuge (Westbury, N.Y.). The supernatant was carefully removed and800 μl of 0.1M sodium phosphate buffer pH 4.8 (activation buffer) wasadded. The beads were then vortexed for 15 seconds and sonicated for 15seconds using a SPER SCIENTIFIC® LTD Ultrasonic Cleaner (Scottsdale,Aris.). The beads were washed an additional time with activation buffer,and resuspended in 200 μl of freshly prepared 5 mg/ml EDC prepared inactivation buffer. The beads were incubated in a rotator for 20 min RTprotected from light. At the end of the EDC step, each bead set waswashed and resuspended with the predetermined concentrations of Aβ₄₀peptides and incubated for lhr RT in a rotator protected from light. Thebead sets were washed and incubated with 1 ml of blocking buffer (PBS,1% (w/v) BSA, 0.02% (w/v) Tween-20) in a rotator for 1 hr RT protectedfrom light. Finally, the concentration of each bead set preparation wasassessed by counting the beads with a hemacytometer. Each bead set wasthen resuspended in blocking buffer at 2×10⁶ beads/ml, and storedprotected from light at 4° C. until ready for use.

Testing of Aβ₄₀ Peptide Coupled Luminex Beads

The presence of Aβ₄₀ peptides which contain the N-terminal epitopes forthe Aβ₄₂N-terminal specific antibodies on the bead surface was confirmedusing surface testing. 50 μl of assay buffer (PBS, 1% (w/v) BSA, 0.05%(w/v) Tween 20, 0.05% (w/v) azide) containing 2500 beads were added toMillipore filter bottom plate wells (Bedford, Mass.). The beads werewashed by placing the plate over a Millipore vacuum manifold (Bedford,Mass.) to remove the liquid and the beads were resuspended in 100 μlwell of PBST wash buffer. Finally, the wash buffer was removed from thewells via vacuum and the beads were incubated with 100 μl well of biotinlabeled 26D6-B2-B3 antibody diluted in assay buffer. The plate wasincubated on a 96-well plate shaker (Lab Line Instruments, Melrose Park,Ill.) at 300 rpm for 30 min RT protected from light. Following theincubation step, the beads were washed 4 times, resuspended in 50μl/well of 1 μg/ml of Phycoerthrin-streptavidin conjugate, and incubatedon a plate shaker for 20 min at RT protected from light. The beads weresubsequently washed 3 times via vacuum filtration using 100 μl/well ofwash buffer and resuspended in 100 μl/well of assay buffer. The MFI ofat least 50 beads/well was measured using a Luminex¹⁰⁰ instrumentobtained from Bio-Rad Laboratories (Hercules, Calif.) running Bioplexmanager 4.1.1 software. The MFI measured on each of the bead sets isshown in FIG. 5A.

Aβ₄₂ Intra-Assay Analysis of Biological Samples

Samples analysis using the intra-assay Luminex based assay was performedby first mixing bead sets that were coupled with anti-C-terminalspecific Aβ₄₂ 565 capture antibodies and bead sets that were coupledwith different concentrations of Aβ₄₀ peptides (shown in FIG. 5A). 50μl/well of the combined mixture of all bead sets at 50,000 beads/mlsuspension prepared in assay buffer was added to each well of a pre-wetfilter bottom 96-well plate. The beads were washed with 100 μl/well ofassay buffer via vacuum filtration. The capture beads were resuspendedin 50 μl of diluted human CSF samples, quality control samples (QC),different concentrations of full length native Aβ₄₂ peptides asreference standards, or different concentrations of modified Aβ₄₂peptide standards in duplicate wells and incubated on a plate shaker for1 hr at RT protected from light. 1.0 μg/ml anti-Aβ₄₂ 26D6 reporterantibody labeled with biotin was added and then incubated on a plateshaker for 0.5 hrs at RT protected from light. Following the incubationstep, the beads were washed 4 times, resuspended in 50 μl/well of 1μg/ml of Phycoerthrin-streptavidin conjugate, and incubated on a plateshaker for 20 min at RT protected from light. Finally, the beads werewashed 4 times and resuspended in 100 μl/well of assay buffer. The MFIof at least 50 beads per well was measured using a Bioplex Luminexinstrument running Bioplex manager 5.1 software (Bio-Rad Laboratories,Hercules, Calif.). Standard curves were generated from the soluble Aβ₄₂peptides or from the signals generated from the bead sets coated withdifferent concentrations of Aβ₄₀ peptides (intra-standard) using aweighted 4-parameter logistic curve fit (FIG. 5B). The concentration ofAβ₄₂ peptides in the CSF or QC samples were calculated from the relevantstandard curve, and are shown in FIG. 5C.

Example 4 Peptide Based Intra-Assay for IGFR1 with Luminex Beads

Below is another example of a peptide intra-assay based assay for thedetection of phosphorylated tyrosine residues, 1162 and 1163, on humanIGF-R1 receptors. Phosphorylated regions of Tau may also be used in thesame manner.

Antibodies and Reference Standards

A custom made IGF1R [PYPY^(1162/1163)] peptide was provided by CambridgeResearch Biochemicals Ltd (UK). Mouse anti-IGF1R capture antibody wasobtained from Calbiochem (San Diego, Calif.) and the rabbitanti-phospho-tyrosine (1162/1163)-IGF1R reporter antibody was purchasedfrom Millipore (Billerica, Mass.). Phycoerythrin label goat anti-rabbitantibody was obtained from Jackson Immunoresearch (West Grove, Pa.).Tween-20, 1-ethyl-3-[3 dimethylaminopropyl] carbodiimide hydrochloride(EDC), sodium azide, IgG free Bovine Serum Albumin (BSA), and sodiumphosphate were purchased from Sigma-Aldrich Corporation (St. Louis,Mo.). Phosphate buffered saline (PBS) was obtained from MediatechIncorporated (Herndon, Va.). Carboxylated Luminex beads were purchasedfrom Bio-Rad Incorporated (Hercules, Calif.). Normal healthy PBMCsamples were obtained from In house donors (BMS, N.J.). The PBMC sampleswere treated with either PBS or 100 ng/ml of purified human IGF1(Genetex Inc, TX) for 10 min at 37° C. to induce phosphorylation of IGFRpresent on the cells. The cells were washed, lysed with a modified RIPAbuffer, and stored at −80° C.

Covalent Coupling of Anti-IGF-R1 Capture Antibody to Luminex Beads

The mouse anti-human IGF1R capture antibody was covalently coupled tothe surface of carboxylated beads using a two-step carbodimideprocedure. The beads were washed by centrifuging 1.25×10⁷ beads for 5min at 14000×g 4° C. in an Eppendorf 5415D centrifuge (Westbury, N.Y.).The supernatant was carefully removed and another 1.25×10⁷ beads weredispensed and centrifuged for 5 min at 14000×g 4° C. The supernatant wasagain carefully removed and re-suspended in 800 μl of 0.1M sodiumphosphate buffer pH 4.8 (activation buffer). The beads were thenvortexed for 15 seconds and sonicated for 15 seconds using a SPERSCIENTIFIC® LTD Ultrasonic Cleaner (Scottsdale, Arix.). The beads werewashed 2 additional times with activation buffer, and resuspended in 200μl of freshly prepared 5 mg/ml EDC prepared in activation buffer. Thebeads were incubated in a rotator for 20 min RT protected from light. Atthe end of the EDC step, the beads were washed and resuspended in 1000μl of 250 μg/ml capture antibody prepared in PBS and incubated for 1 hrRT in a rotator protected from light. The beads were washed andincubated with 1 ml of blocking buffer (PBS, 1% (w/v) BSA, 0.02% (w/v)Tween-20) in a rotator for 1 hr RT protected from light. Finally, thebeads were counted with a hemacytometer, resuspended in blocking bufferat 2×10⁶ beads/ml, and stored protected from light at 4° C. until readyfor use.

Testing of Covalent Coupled IGF-R1 Capture Antibodies to Luminex Beads

The presence of capture antibody on the bead surface was confirmed usingsurface testing. 50 μl of assay buffer (PBS, 1% (w/v) BSA, 0.05% (w/v)Tween 20, 0.05% (w/v) azide) containing 2500 beads were added toMillipore filter bottom plate wells (Bedford, Mass.). The beads werewashed by placing the plate over a Millipore vacuum manifold (Bedford,Mass.) to remove the liquid and the beads were resuspended in 100μl/well of PBST wash buffer. Finally, the wash buffer was removed fromthe wells via vacuum and the beads were incubated with 100 μl/well ofPE-GAM diluted 1/100 in assay buffer. The plate was incubated on a96-well plate shaker (Lab Line Instruments, Melrose Park, Ill.) at 300rpm for 30 min RT protected from light. The beads were subsequentlywashed 3 times via vacuum filtration using 100 μl/well of wash bufferand resuspended in 100 μl/well of assay buffer. The MFI of at least 50beads/well was measured using a Luminex¹⁰⁰ instrument obtained fromBio-Rad Laboratories (Hercules, Calif.) running Bioplex manager 4.1.1software. An MFI of at least 20,000 was used to confirm the presence ofusable quantities of antibody on the bead surface.

Covalent Coupling of Intra-assay IGF-R1 Peptide Standards to LuminexBeads

IGF1R [PYPY1162/1163] reference standard peptides were prepared byreconstituting the lyophilized peptide in 2.5 ml PBS to give a finalconcentration of 10 mg/ml. Initial 10 fold dilution with subsequent10-fold were made using diluent buffer (PBS, 1% (w/v) BSA, 0.02% (w/v)Tween-20). The phospho-IGF1R peptide was covalently coupled to thesurface of four different sets of carboxylated beads at four differentconcentrations using a two-step carbodimide procedure. The bead setswere washed by centrifuging 1.25×10⁷ beads for 5 min at 14000×g 4° C. inan Eppendorf 5415D centrifuge (Westbury, N.Y.). The supernatant wascarefully removed and 800 μl of 0.1M sodium phosphate buffer pH 4.8(activation buffer) was added. The beads were then vortexed for 15seconds and sonicated for 15 seconds using a SPER SCIENTIFIC® LTDUltrasonic Cleaner (Scottsdale, Aris.). The beads were washed anadditional time with activation buffer, and resuspended 200 μl offreshly prepared 5 mg/ml EDC prepared in activation buffer. The beadswere incubated in a rotator for 20 min RT protected from light. At theend of the EDC step, each beads were washed and resuspended withindividual concentration of 1000, 100, 10 and 1 ng/ml of phospho-IGF1Rpeptide made from a 10-fold serial dilutions of 10 mg/ml prepared inPBS. The beads were then incubated for 1 hr RT in a rotator protectedfrom light. The beads were washed and incubated with 1 ml of blockingbuffer (PBS, 1% (w/v) BSA, 0.02% (w/v) Tween-20) in a rotator for 1 hrRT protected from light. Finally, the beads were counted with ahemacytometer, resuspended in blocking buffer at 2×10⁶ beads/ml, andstored protected from light at 4° C. until ready for use.

Testing of IGF-R1 Peptide Coupled Luminex Beads

The presence of phospho-IGF1R peptide on the bead surface was confirmedusing surface testing. 50 μl of assay buffer (PBS, 1% (w/v) BSA, 0.05%(w/v) Tween 20, 0.05% (w/v) azide) containing 2500 beads were added toMillipore filter bottom plate wells (Bedford, Mass.). The beads werewashed by placing the plate over a Millipore vacuum manifold (Bedford,Mass.) to remove the liquid and the beads were resuspended in 100μl/well of PBST wash buffer. Finally, the wash buffer was removed fromthe wells via vacuum and the beads were incubated with 100 μl/well ofrabbit anti-phospho-IGF-R1 antibody diluted in assay buffer. The platewas incubated on a 96-well plate shaker (Lab Line Instruments, MelrosePark, Ill.) at 300 rpm for 30 min RT protected from light. Following theincubation step, the beads were washed 4 times, resuspended in 50μl/well of 1 μg/ml of phycoerthrin labeled goat anti-rabbit IgGconjugate, and incubated on a plate shaker for 20 min at RT protectedfrom light. The beads were subsequently washed 3 times via vacuumfiltration using 100 μl/well of wash buffer and resuspended in 100μl/well of assay buffer. The MFI of at least 50 beads/well was measuredusing a Luminex¹⁰⁰ instrument obtained from Bio-Rad Laboratories(Hercules, Calif.) running Bioplex manager 4.1.1 software. phospho-IGF1Rpeptide,

IGF-R1 Intra-Assay Analysis of Biological Samples

Sample analysis using the Luminex based assay was performed by adding 50μl of a 50,000 beads/ml of capture antibody suspension prepared in assaybuffer to each well of a pre-wet filter bottom 96-well plate. The beadswere washed with 100 μl/well of assay buffer via vacuum filtration. Thecapture beads were resuspended in 50 μl of diluted samples, or qualitycontrol samples (QC) in duplicate wells and incubated on a plate shakerfor 1 hr at RT protected from light. The beads were washed 4 times,resuspended in 50 μl of a 50,000 beads/ml of the four different beadsets of peptide mentioned in section 2.2.3. The beads were filtered,resuspended in 50 μl/well of 1.0 μg/ml anti-phospho IGF1R reporterantibody, and incubated on a plate shaker for 0.5 hrs at RT protectedfrom light. Following the incubation step, the beads were washed 4times, resuspended in 50 μl/well of 1 μg/ml of PE-Goat anti rabbit IgG,and incubated on a plate shaker for 20 min at RT protected from light.Finally, the beads were washed 4 times and resuspended in 100 μl/well ofassay buffer. The MFI of at least 50 beads per well was measured using aBioplex Luminex instrument running Bioplex manager 4.1 software (Bio-RadLaboratories, Hercules, Calif.). The standard curve generated from thephospho-IGF-R1 intra-standard curve is shown in FIG. 6A. Thephospho-IGF1R concentration in each of the PBMC lysate samples using theintra-assay 4-parameter logistic curve fit is shown in FIG. 6B.

Example 5 Characterization of Aβ Peptides Peptides

All peptides were received as lyophilized powder. Modified peptides wereobtained from GenScript (GS) and Anaspec (AN). These peptides weresynthesized using solid phase methods known to those skilled in the art(See, for example, Barany, G. et al., The Peptides: Analysis, Synthesis,Biology—Special Methods in Peptide Synthesis, Part A, Vol. 2, pp. 3-284,Gross, E. et al., eds., Academic Press, New York, publ. (1980); and inStewart, J. M. et al., Solid-Phase Peptide Synthesis, 2nd Edition,Pierce Chemical Co., Rockford, Ill., publ. (1984)).

GS1-6 and AN7 peptides were received and reconstituted in ddH20 to aconcentration of 1 mg/ml. These were then aliquoted out into 100 μlaliquots in the1.4 ml blank tubes PP, round Matrix (Thermo Scientific,cat#4249, lot 1030509). The full length Aβ₄₂ peptide was purchase fromMSD (lot # T03080X1). This peptide was diluted in DMSO to make asolution of 0.1 mg/ml. This was then aliquoted out in 100 μl aliquots inthe 1.4 ml blank tubes. These peptides were kept frozen at −70° C. forstorage. FIG. 13 shows the structures of the polyethylene glycol spacersincorporated into the modified Aβ₍₁₋₄₂₎ peptides.

TABLE 5 List of Aβ Peptides % Purity GS# Peptide Sequence (HPLC) MW GS 1DAEFRHDSGYEVHHQK-{PEG(20-atoms}3- 95 3406.71 GGVVIA (SEQ ID NO: 12) GS 2DAEFRHDSGYEVHHQK-{PEG(9-atoms}6- 95 3553.02 MVGGVVIA (SEQ ID NO: 13)GS 3 DAEFRHDSGYEVHHQK-PEG(9-ATOMS)5- 95 3691.15IGLMVGGVVIA (SEQ ID NO: 14) GS 4 DAEFRHDSGYEVHHQKEERPIGLMVGGVVIA 963476.91 (SEQ ID NO: 6) GS 5 DAEFRHDSGYEVHHQKDREPNRIGLMVGGV 97 3733.17VIA (SEQ ID NO: 8) GS 6 DAEFRHDSGYEVHHQKIGLMVGGVVIA 98 2965.37(SEQ ID NO: 4) AN 7 DAEFRHDSGYEVHMVGGVVIA 90 2288.49 (SEQ ID NO: 2) FLDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAI IGLMVGGVVIADAEFRHDSGYEVHMVGGVVIA(SEQ ID NO: 19)

Dynamic Light Scattering

The novel peptides (GenScript) were received as dry powders, and twosets of stock samples were prepared by weighing small amounts (˜0.5 mgeach) in 1.8 ml polypropylene tubes and dissolving in a simple phosphatebuffer (10 mM Na₂HPO₄, pH 7.4, 10 mM NaCl, prepared in 99.9% D₂O;filtered through 0.2 μm filter). One set was prepared at 1.0 mg/ml, thesecond set was prepared at 0.10 mg/ml, and stored at room temperature.Peptides were dissolved by vortexing for 1 min, and centrifuged in amicrocentrifuge for 5 min at 14,000 rpm, at room temperature. For Aβ₄₂samples (MSD), vials containing 0.1 mg peptide were suspended in 1.0 mlbuffer, and treated as above. A volume (200 μl) of each centrifuged 1.0mg/ml stock was transferred to a 0.5 ml polypropylene tube, and providedfor NMR analysis.

The absorbance spectra of all samples were recorded (220-750 nm) using aNANODROP® ND-1000 instrument, and blanked against the buffer. Dynamiclight scattering analysis was conducted on the peptides in a 384 wellpolystyrene plate (CORNING®, type 3540) with a Wyatt DLS DynaPro platereader. Data collection and analysis was completed using software fromthe manufacturer (DYNAMICS, versions 7.0.0.94 and 7.0.1.12). Eachpeptide was evaluated in triplicate with 30 μl loaded per well, andoverlaid with 5 μl mineral oil, to minimize evaporation. Buffer blankswere also included in the analysis for comparison. After loading theplate, a transparent adhesive sealing tape cover was applied to theplate and the plate was centrifuged for 2 min at 1000 rpm. Each sampleon the plate was read 30 times, 5 sec per acquisition, maintaining atemperature of 25° C. Data sets were collected on both the 1.0 mg/ml and0.10 mg/ml samples over the course of 27 days. The adhesive cover wasremoved to read the samples in the plate reader, and used to cover theplate between readings over the 27 day time course.

FIGS. 7A through 7F show dynamic light scattering data. In FIG. 7, thefull length Aβ₄₂ peptide was subjected to DLS analysis and compared tofour representative modified peptides. The full length peptide was shownto be aggregated with results that calculated the radius of thedifferent aggregate species to be between 82 and 231 nm in length. Thisadds up to large molecular weight aggregates in the solution. However,the modified peptides remain monomeric as determined by both molecularweights as well as radius. GS#3 was contrasted with the other peptidesbecause it did retain some aggregation (FIG. 7E). These resultsdemonstrate that the modified peptides which are lacking an aggregationdomain, do in fact remain monomeric in solution. FIG. 7 a represents theraw data accumulated for all five peptides. Shown is the intensityautocorrelation versus time. The higher the intensity autocorrelationnumber is represents larger diameters of the peptides in solution. FIGS.7 b-7 f depict the percent mass versus the radius of the peptides.Monomeric peptides would be represented by a larger percent mass at thesmaller radius size. The larger aggregated peptides have smaller percentmasses at a higher radius size.

TABLE 6 Summary of DLS data Radius % Mw-R % % Item (nm) Pd (kDa)Intensity Mass GS#1 Peak1 1.2 4.6 5.0 6.3 99.9 Peak 2 48.4 3.9 29512.093.7 0.1 GS#2 Peak 1 0.9 4.3 3.0 1.6 98.0 Peak 2 4.6 1.9 117.0 3.4 1.8Peak 3 36.9 3.8 15580.0 76.1 0.1 Peak 4 172.0 4.5 572266.0 18.9 0.1 GS#3Peak 1 4.5 0.0 119.0 0.4 57.9 Peak 2 15.7 4.5 2101.0 4.8 19.7 Peak 361.6 4.5 51794.0 94.8 22.4 GS#4 Peak 1 1.3 3.2 6.0 3.8 97.7 Peak 2 6.00.0 225.0 0.6 0.2 Peak 3 27.0 4.5 7527.0 5.2 0.0 Peak 4 127.0 4.3283287.0 25.1 0.2 FL Aβ1-42 Peak 1 82.9 4.4 103812.0 50.4 26.1 Peak 2231.0 3.2 1149570.0 49.6 73.9

Circular Dichroism

Circular dichroism (CD) spectra were collected using an Aviv Model 202Circular dichroism spectrometer. Samples were pipetted in 1 mm pathlength quartz cuvettes, and scanned from 260-185 nm. Parameters forcollecting data include spectral bandwidth of 1.00 nm, a step size of1.0 nm, an averaging time of 20 sec per point, and temperature set at25° C. Samples were stored in the quartz cuvettes at room temperature,and scanned over a time course of 23 days. Raw data was corrected forblank buffer contributions, and presented in the format of mdeg versuswavelength.

FIG. 8 shows the circular dichroism analysis. Circular dichroismanalysis was performed on the full length Aβ₄₂ and four representativepeptides to determine the order and secondary structures of thesepeptides. As shown in FIG. 8, the full length peptide shows more of anordered structure than the other peptides. The four representativepeptides contain an unordered CD spectra when compared to the fulllength peptide. These data suggest that the modified peptides do notaggregate or form any higher order structures unlike that of the fulllength peptide.

MSD ELISA

Biotin labeled anti-C-terminal Aβ₄₂ antibody (565-20 μg/ml) wasimmobilized to a 96 well MSD streptavidin coated plate by adding 30μl/well. The plate was then covered and incubated overnight at 25° C.,shaking at 500 rpm. The plates were then washed 3× with 300 μl/well ofwash buffer (R & D system cat# WA126). After washing, 225 μl/well ofblocking buffer (1%BSA+0.1% Tween-20 in PBS) was added to the plate andincubated for 30 minutes at room temperature, shaking at 500 rpm. Oncethe blocking step was done, the plates were washed as describedpreviously. Reference standard Aβ₄₂ peptides (full length or modifiedpeptides) were added to the wells. Aβ₄₂ peptides were captured by theimmobilized capture antibody. A second ruthenium tagged (Ru) antibody tothe N-terminus of Aβ₄₂ (26D6) was added thereby completing the sandwich.The complex was detected using an MSD sector 6000 instrument using ECL.The raw fluorescence units (RFU) measured by the instrument were fit toa 4-parameter logistic model to create a standard curve.

FIG. 9 shows peptide stability data. The full length Aβ₄₂ and sevenmodified peptides were subjected to stability studies at differenttemperatures for up to 40 days (FIGS. 9A-9F). These peptides were keptat 4° C., 25° C., and 37° C. for the specified length of time and thenfrozen until the assay was run. Peptides were then collected and run onan MSD Elisa format. Results are shown as a percent of the baselinesignal for each peptide measured at time zero. Indeed, short termincubation at 37° C. has been used to estimate the long term stabilityof molecules at 4° C. and room temperature (Anderson et al., ClinicalChemistry, 37(3): 398-402 (1991)). Therefore, incubation of the peptidesat 37° C. for short term analysis can be used to assess the long termstability of the peptides. As shown in FIG. 9, the full length Aβ₄₂peptide signal drops dramatically starting at 16 hr incubation at 37° C.In contrast, the modified peptides remain stable at all temperaturesmeasured. This data would suggest that these modified peptides are morestable than the full length Aβ₄₂.

FIGS. 10A-10I show the full length Aβ₄₂ and seven modified peptides weresubjected to stability studies at different temperatures for up to 60days. These peptides were kept at 25° C. and 37° C. for the specifiedlength of time and then frozen until the assay was run. Peptides werethen collected and run on an MSD Elisa format. Results are shown as astandard curve, plotting signal versus peptide concentration. As shownin FIG. 10, the full length peptide gave decreasing standard curves at25° C. and 37° C. In comparison, all seven peptides analyzed gavesimilar curves across all time points and temperature ranges (only 37°C. shown). The results suggest that the modified peptides will give amore consistent signal than the full length Aβ₄₂peptide when used as astandard for assay reproducibility.

FIG. 11 shows the standard curve analysis of full length versus modifiedpeptides. Full length Aβ₄₂ and seven modified peptides were diluted outfor an eight point calibration curve and ran on the MSD Elisa format. Asshown in FIG. 11, all of the peptides produced almost identical standardcurves. This result suggests that the modified peptides can be used inthe place of the full length peptide for the calculation of Aβ in samplematrixes.

FIG. 12 shows a CSF sample analysis using full length versus modifiedpeptides. Full length Aβ₄₂and seven modified peptides were diluted outfor an eight point calibration curve and ran on the MSD Elisa formatwith 13 CSF samples. The concentration of Aβ in each CSF sample wascalculated based on the standard curve for each peptide. As shown inFIG. 12, the back calculated Aβ concentrations in the CSF samples wereequivalent and was independent of which Aβ peptide used for the standardcurve. This suggests that these modified peptides can be used in placeof the full length Aβ₄₂ peptide in these assays without affecting thecalculations of the Aβ levels.

1. A composition comprising an N-terminal immunoreactive region, a C-terminal immunoreactive region and a linker region.
 2. A composition comprising an N-terminal immunoreactive region, a C-terminal immunoreactive region and a linker region, wherein the composition is used as a reference standard in an immunoassay.
 3. The composition of claim 2 wherein the immunoassay is selected from the group consisting of a sandwich immunoassay, a single antibody assay, a double sandwich immunoassay and a competition assay.
 4. The composition of claim 1, wherein the N-terminal immunoreactive region binds Aβ₄₂.
 5. The composition of claim 1, wherein the C-terminal immunoreactive region binds Aβ₄₂.
 6. The composition of claim 1, wherein the linker region is a non-immunoreactive domain.
 7. The composition of claim 6, wherein the linker region comprises a linker selected from the group consisting of polyethylene glycol, a glutamine residue, an alanine residue, a lysine residue, a lipid, a globular protein, a nucleic acid and an alkyl chain.
 8. An isolated modified peptide molecule having an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23, 24, 25, 26 and
 27. 9. A method of measuring the quantity of an analyte in a biological sample, the method comprising: (a) attaching a reference standard to at least two beads thereby forming a first bead set and a second bead set, wherein the reference standard comprises an epitope recognized by a first detection antibody and wherein each bead set comprises a different concentration of the reference standard; (b) attaching a capture antibody specific to the analyte to a third bead set; (c) mixing all of the bead sets together to form a suspension array; (d) applying the biological sample to the suspension array whereby the analyte binds to the capture antibody on the third bead set; (e) adding a first detection antibody to the suspension array, wherein the first detection antibody binds the reference standard and analyte bound to the capture antibody; (f) measuring a first signal from the first detection antibody bound to the reference standard in the first bead set; (g) measuring a second signal from the first detection antibody bound to the reference standard in the second bead set; (h) generating a standard curve based upon the first and second signals; and (i) quantitating the amount of the analyte in the third bead set by measuring a third signal from the first detection antibody and comparing the third signal to the first and second signal measurements on the standard curve.
 10. The method of claim 9, wherein the reference standard comprises the composition of claim
 1. 11. The method of claim 9, wherein the biological sample is selected from the group consisting of blood, serum, plasma, peripheral blood mononuclear cells, peripheral blood lymphocytes, tissue, cerebrospinal fluid and cells.
 12. The method of claim 9, wherein the analyte is selected from the group consisting of Aβ₄₂, Aβ₄₀, Aβ₃₈, tau and insulin growth factor receptor
 1. 13. The method of claim 9, wherein the method is performed in a multi-well plate, nitrocellulose filter, glass fiber or on a glass slide.
 14. The method of claim 9, wherein the first signal and second signal is a signal selected from the group consisting of phycoerythrin, alexa 532, streptavidin-phycoerythrin and streptavidin-Alexa
 532. 15. A kit for conducting an immunoassay to detect Aβ₄₂ peptide, the kit comprising a composition of claim
 1. 16. The composition of claim 2, wherein the N-terminal immunoreactive region binds Aβ₄₂.
 17. The composition of claim 2, wherein the C-terminal immunoreactive region binds Aβ₄₂.
 18. The composition of claim 2, wherein the linker region is a non-immunoreactive domain.
 19. The method of claim 9, wherein the reference standard comprises the composition of claim
 2. 20. The method of claim 9, wherein the reference standard comprises the composition of claim
 8. 21. A kit for conducting an immunoassay to detect Aβ₄₂ peptide, the kit comprising a composition of claim
 2. 22. A kit for conducting an immunoassay to detect Aβ₄₂ peptide, the kit comprising a composition of claim
 8. 